The major objectives of this Project are: 1, to determine the 3- dimensional (3-D) distribution of Ca2+-channels and of the organelles and membranes containing these channels, in vascular smooth and cardiac muscles and 2, to characterize the molecular topology of the smooth muscle inositol 1,4,5-triphosphate (InsP3) receptor, and 3, to determine the mechanisms targeting the Ca2+-ATPase and the (Na/K+)-ATPase to their, respectively, intracellular and plasmalemmal functional all sites. The Ca2+-channels to be studied are the ryanodine- and InsP3-receptors of the sarcoplasmic reticulum (SR) and the dihydropyridine (DHP)-receptor (L-channel) of the plasma membrane. A functionally important objective is to determine whether and at which sites these receptor-channels are colocalized at surface couplings of smooth and cardiac muscle, cardiac triads and other regions involved in excitation-contraction coupling. A 3-D reconstruction of the cardiac T-tubular network will be obtained and its DHP (L-channel) content will be determined. Fluorescent antibodies and fluorescent ligands will be used, in conjunction with laser scanning confocal microscopy and/or immunoelectron microscopy for localizing ion transporting proteins. These studies are aimed at a better understanding of the mechanisms of excitation-contraction (E-C) coupling in cardiac and in vascular smooth muscle, and of the possible influence of the spatial distribution of DHP- receptor Ca2+ channels on intercellular conduction and on cardiac arrhythmias. The aims of this Project are intimately related to those of Project 5, and the imaging approach, confocal microscopy, is an essential complement to the molecular biological studies, conducted in Project 4 for determining the mechanisms of transporting enzyme targeting.